Apoptosis and Oncotic Necrosis:The final common pathways of liver injury

Hartmut Jaeschke

Department of Pharmacology, Toxicology & Therapeutics

University of Kansas Medical CenterKansas City, KS

Fibrosis: Response to Cell Injury

Rockey & Friedman, 2006

Chronic Cell Injury: Apoptosis and Oncotic Necrosis

• cellular condensation (cell shrinkage)

• membrane blebbing, but no loss of integrity

• aggregation of chromatin at the nuclear membrane

• formation of membrane bound vesicles (apoptotic bodies)

• no disintegration of organelles; organelles remain intact

• swelling of the cell and lysis

• loss of membrane integrity

• flocculation of chromatin

• no vesicle formation, complete lysis

• disintegration (swelling) of cell organelles

Morphology

Apoptosis Oncotic Necrosis

Hepatocellular Apoptosis

H & E

Galactosamine(500 mg/kg; 6 hr)

Hepatocellular Oncotic Necrosis and Inflammation

H & E

Bile Duct Ligation3 days

Apoptotic Cell Death: Caspases

Proenzyme (zymogen)

prodomain large subunit linker domain small subunit

Peptidase1 496

DED DED Pro-Caspase 8Initiator Caspase

Death effector domainDED

Peptidase1 277 Pro-Caspase 3

Effector Caspase

Cysteine-dependentaspartase domainPeptidase

PeptidaseCARD1 416 Pro-Caspase 9

Initiator Caspase

Caspase recruitment domainCARD

Activation of CaspasesProenzyme(zymogen)

Active Caspase(tetramer)

prodomain large subunit linker domain small subunit

Activation of CaspasesProenzyme(zymogen)

Active Caspase(tetramer)

prodomain large subunit linker domain small subunit

0 20 40 90 120 Time (min)

p32

p11Fas-AbJo-2

Proenzyme

Active Fragment

0

2 0 0

4 0 0

6 0 0

Activation of Caspases

0 20 40 90 120 Time (min)

p32

p11

(ΔF/min/mg protein)

600

400

200

0

*

*

*

DDDD DD

FAS-L FAS-L

DDDDDD

FAD

DFA

DD

FAD

DFA

DD

Cas8

FAS-L

DDDDDD

FAD

DFA

DD

FAD

DFA

DD

Cas10

Cas

p10

Casp 6

Casp

8Bid

BaxBak

C

C

C

C

C

C

C

CC

C

C

CC

C

Casp 7Casp 3A

paf1

Apa

f1

Cas

p9

Cas

p9

C

CC

C

CAD

Endo

GEn

doG

CAD

Endo

G

AIF

AIF

Dia

blo

Diablo

C

Bak Bak

Bax

Bax

CaspaseSubstrates

CAD

CARD

C

C

CARD

DISC

FAS-LFAS-L

dATP

dATP

Fas-mediated Apoptosis Signaling in Hepatocytes

CAD ICADCAD

Bid

Bcl-2

Bak

C

C

CC

C

C

C

C

C

C

C

C Casp 3

C

C

CC

EndoG EndoG

EndoG

EndoG

AIF

Diablo

C

Caspase 3 SubstratesAIF

cIAPDiablo

Intrinsic Pathway of Apoptosis

Ca

Ca

Ca

Ca

AIF

Diablo

Diablo C

AIF

C

C

C

C

C

C

C C

CC

C

C

BaxBakBax

Bak

AIFDiablo

CaCa

Ca

Ca

CaCa

CaCa

CaCa

PTP

Bcl-XL

Bcl-2 Bax

Bax

Bax

28S

eIF2α

PTP

Cathepsin

C

Apa

f1CARD

Apaf1CAR

D

Apaf1 CARD

Apaf1

CARDCasp 9

Casp

9Casp

9

C

CC

BaxBakBax

Bak

CaCaCa

Ca

Casp 9

C C

m-Calpain

Casp 12

p53Bax

Caspase Targets in ApoptosisCasp10

Casp 6

Casp 8

Casp 7

Casp 3

Apa

f1

Apa

f1

Cas

p9

Cas

p9

CARDC

CARD

CARD Casp 2

MDM2Fodrin

Prese-nelin2 Gelsolin

Actin Lamin A

DNA fragmentation andchromatin condensation

CytoskeletonCell shape and membrane

blebbing

CAD

Trans-glutaminase

Topo-isomerase

PARPDNA-PKEndoGICAD

Keratin-18

Gas2

Cell cycle and other

p21

FAK

hnRNP

β-catenin

NuMASREB1

Calpastatin

EMAP II

Rock-1

DNA Fragmentation

1 2 3 4 5 6 7 8 9 10 11 12 13

MW Controls G/ET

0

200

400

600

800

1000

(% Vmax)

00 20 40 90 120

600

400

200

800

1000 *

*

*

Time (min)

DNA LadderAnti-Histone ELISA

CADCAD

DNA Strand Breaks: TUNEL Assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling

GalactosamineGalactosamine (500 mg/kg; 6 hr)(500 mg/kg; 6 hr)

Characteristic Features of Oncotic Necrosis

• Morphology: cell swelling, cell contents release, karyolysis

Mechanism is dependent on the insult

Oncotic Necrosis: Acetaminophen- induced Hepatotoxicity

Cell Swelling, Karyolysis

Cell Contents Release : Plasma ALT > 3000 U/L)

DNA Strandbreaks during OncoticNecrosis

TUNEL Assay

CV

4 h APAP 6 h APAP

DNA Strandbreaks: Oncotic Necrosis vs Apoptosis

TUNEL Assay

CV

6 h Gal/ET 6 h APAP

DNA Fragmentation:Apoptosis vs Oncotic Necrosis

1 2 3 4 5 6 7 8 9 10 11 12 13

MW Controls G/ET

DNA LadderAnti-Histone ELISA

APAP

DN

A F

ragm

enta

tion

(% c

ontro

l)

0

500

1000

1500

2000

C G/E G/E ZVAD

AAP AAP ZVAD

AAP GSH

*

* *#

#

1 2 3 4 5 6 7 8 9

0 0.5 1 2 3 4 6 G/E

Acetaminophen (h)

p32

p11

Caspase-3 Processing during Acetaminophen Toxicity

Lawson et al., Toxicol Appl Pharmacol 156: 179-86, 1999 Gujral et al., Toxicol Sci 67: 322-8, 2002

BaxBak

C

C

C

C

C

C

C

CC

C

C

CC

C

C

CC

C

EndoG

Endo

GEn

doG

EndoG

Endo

G

EndoG

AIF

AIF

Dia

blo

Diablo

C

Bak Bak

Bax

Bax

AIF

C

DiabloAIF

EndoG

DNA Fragmentation: Nuclear Translocation of Mitochondrial Intermembrane Proteins

Diablo

C C

Nucleus

Mitochondria

Control

5 mM AAP, 6 h

Endonuclease G

Bajt et al., Toxicol Sci 94: 217-225,2006

Cytochrome c

Bax

BidtBid

Contr

olAP

AP 2

h

APAP

4 h

Contr

olAP

AP 2

hAP

AP 4

h

Mitochondria Cytosol

APAP-induced Bax and tBid Translocation to Mitochondria

APAP Hepatotoxicity in Bax Gene Knockout Mice

Pla

sma

ALT

(U/L

)

0

1000

2000

3000

4000

5000

6000

7000

*

WT Bax-/-

Bajt et al., J Pharmacol Exp Therap, in press, 2007

APAP Hepatotoxicity in Bax Gene Knockout Mice

Pla

sma

ALT

(U/L

)

0

1000

2000

3000

4000

5000

6000

7000

*

WT Bax-/-

Bajt et al., J Pharmacol Exp Therap, in press, 2007

WT

Bax-/-

NAPQI APAPGSH ↓

Protein Arylation

P4501.

2.

Bax

Cyt c / SmacAIF Endonuclease G

ONOO-

Nucleus

DNA-Strandbreaks

ATP ↓

CaspaseActivation

ATP ↓

PARP Activation

DNA Repair

DNA-Fragmentation

Chromatin Condensation

NAD+ Depletion

ATP ↓ ⇐ MPT

?Mechanism of Mechanism of APAP ToxicityAPAP Toxicity

Toxicol Sci 89: 31-41, 2006

O2 –NO

Bax MPT

Protein Nitration

Apoptosis vs Oncotic Necrosis

Initiation Execution

Apoptosis ApoptosisATP high

Apoptosis Secondary NecrosisATP low

Oncosis Oncotic NecrosisATP low

OncosisATP high Secondary Apoptosis ?

Same process or replaced by different mode of cell death?

Cell Death Connection to Fibrosis

Apoptotic Bodies

PhagocytosisHSC

KC

TGFβ-1

TNF-α

Canbay et al., Lab Invest 83:655,2003

Cell Death Connection to Fibrosis

Phagocytosis

HSC

KC

TGFβ-1

TNF-α

HMGB1

Cell Debris

HSC

KC

TLR4TGFβ-1

TNF-α

Fibrosis: Response to Cell Injury

Rockey & Friedman, 2006

Apoptosis

Oncotic Necrosis